Coupled with temporally regulated translation, subcellular localization of messenger RNA is an important mechanism of post-transcriptional regulation of gene expression. Incorrect mRNA localization disrupts asymmetric cell division, long-term memory formation, as well as the establishment of basic body axes. Consequently, failure to localize correctly can have catastrophic effects on synaptic plasticity underlying learning and memory, and may lead to diseases such as the Fragile X syndrome and spinal muscular atrophy. After nuclear transcription, mRNA is trafficked to specific destinations in the cytoplasm as mRNA: protein (mRNP) complexes. Efficient transport of mRNAs requires highly orchestrated interactions between nuclear and cytoplasmic proteins and the transcript. Deciphering the spatial and temporal organization of these dynamic and sometimes fleeting interactions requires direct observation in vivo. Using high resolution fluorescence imaging, our long-term goal is to describe the composition and functional role of important large mRNP complexes in vivo. To that end, the objective of this application is to examine the spatio-temporal requirements of trans-acting factors during mRNA transport in Drosophila melanogaster oocytes. The central hypothesis of the application is that the posterior pole determinant, oskar mRNA, interacts with several proteins during the dynamic process of mRNA transport in a multi-step mechanism. The rationale for the proposed research is that by combining advanced fluorescent probes and microscopic techniques, we will provide a new avenue to study dynamic molecular interactions in living cells. In order to test how oskar mRNA is processed during Drosophila oogenesis, the following two specific aims will be pursued: 1. Characterize the dynamic steps of oskar mRNA transport during mid-oogenesis. 2. Identify the spatio-temporal relationship of the trans-acting proteins and oskar mRNA during its transport at mid-oogenesis. The proposed work is innovative because it will allow for the first time a real time view of the molecular dynamics of endogenous mRNAs and associating proteins by taking advantage of advanced fluorescence imaging techniques. The complex composition of mRNPs will be resolved in space and time. These studies will improve the models of RNA transport and localization and contribute to a better understanding of the pathogenesis of diseases caused by genetic errors that affect these processes.